15 research outputs found
Distinct axo-protective and axo-destructive roles for Schwann cells after injury in a novel compartmentalised mouse myelinating coculture system.
Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures have been an important technique over the last four decades in understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and rat DRG neurons are commonplace, there are no established protocols in the field describing the use of mouse SCs with mouse DRG neurons in dissociated myelinating cocultures. There is a great need for such a protocol as this would allow the use of cells from many different transgenic mouse lines. Here we describe a protocol to coculture dissociated mouse SCs and DRG neurons and induce robust myelination. Use of microfluidic chambers permits fluidic isolation for drug treatments, allows cultures to be axotomised to study injury responses, and cells can readily be transfected with lentiviruses to permit live imaging. We used this model to quantify the rate of degeneration after traumatic axotomy in the presence and absence of myelinating SCs and axon aligned SCs that were not induced to myelinate. We find that SCs, irrespective of myelination status, are axoprotective and delay axon degeneration early on. At later time points after injury, we use live imaging of cocultures to show that once axonal degeneration has commenced SCs break up, ingest, and clear axonal debris
SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice
Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.CM was funded by a Medical Research Council (UK) studentship (2251399). PA-F (206634/Z/17/Z), AL (210904/Z/18/Z), CC (220027/Z/19/Z), RB (203151/Z/16/Z) and MC (220906/Z/20/Z) were funded by the Wellcome Trust (UK). BS was supported by a Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (109408/Z/15/Z). KM was funded by the National Institute of Neurological Disorders and Stroke Awards (R01NS079445). JG-S was funded by a Miguel Servet Fellowship (CP22/00078) from the Instituto de Salud Carlos III and the Millennium Nucleus for the Study of Pain (MiNuSPain), Santiago, Chile. HC was funded by the Spanish “Ministerio de Economía y Competitividad” (BFU2016-75864R and PID2019-109762RB-I00), ISABIAL (UGP18-257 and UGP-2019-128), and Generalitat Valenciana (PROMETEO 2018/114). Y-PH and C-YC were funded by Academia Sinica, AS-IA-106-L04 to Y-PH.Peer reviewe
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Axon Degeneration and the Schwann Cell Early Injury Response: A Study in Mouse and Zebrafish
After peripheral nervous system injury, axons degenerate through a process termed Wallerian degeneration and Schwann cells transform into a repair phenotype. Axon degeneration is regulated by a signalling pathway controlled by the pro-degenerative axon death molecule sterile-alpha and toll/interleukin 1 receptor motif containing protein 1 (SARM1). Meanwhile, Schwann cells activate a distinct transcriptional response, digest myelin using myelinophagy, attract macrophages, and support the survival of damaged neurons and their growth and guidance to their target. The early Schwann cell injury response prior to and around the timing of axon degeneration has, however, not been investigated in great detail, and the identity of an axonal injury signal that induces this Schwann cell injury response remains elusive.
In order to investigate axon Schwann cell interactions in vitro, I developed a novel compartmentalised dissociated dorsal root ganglion neuron and Schwann cell coculture model. I show that in this model, Schwann cells are initially axo-protective, as their presence delays degeneration, irrespective of their myelination status. In later phases after injury, they are then axo-destructive and fragment and phagocytose axons. To further investigate the role of Schwann cells after injury, I then characterised an in vivo model of peripheral nervous system injury in larval zebrafish. I describe the rate of axon degeneration after laser axotomy of the peripheral lateral line nerve in wildtype, as well as in sarm1 mutant animals. I show a characteristic delay in axon degeneration in sarm1 mutant zebrafish, while their myelination is normal. I then performed cell specific reexpression experiments with human SARM1 and show that neuronal SARM1 is sufficient to rescue axon degeneration. In a more traditional mouse model of peripheral nervous system injury, I performed a bulk RNA sequencing study of the distal tibial nerve after cut injury at the sciatic notch at early timepoints after injury in both Wildtype and Sarm1 knockout mice. Previous studies have mainly focussed on late timepoints, but I show a much earlier induction of the Schwann cell injury response, prior to myelinated axon degeneration. I then further investigated the timing of unmyelinated axon degeneration and show that these degenerate before myelinated axons do, at timepoints that correspond to the induction of the early Schwann cell injury response. I further show that Schwann cells likely do not express SARM1 and are insensitive to SARM1 activation, suggesting the delayed degeneration in Sarm1 knockout mice is solely due to the axonal absence of Sarm1.
Overall, this thesis details novel methods to investigate peripheral nervous system injury and provides novel insights into early events after injury | both in Schwann cells and axons. Results provide insights into Schwann cell axon interactions after injury, and highlight key differences between myelinated and unmyelinated axons that warrant further investigation
The Schwann cell early injury response in mouse and zebrafish
Trabajo presentado al Seminario de Unidad Neurobiología Molecular y Neuropatología del Instituto de Neurociencias, celebrado online el 20 de julio de 2021.Peer reviewe
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Glial plasticity in the zebrafish central nervous system.
Glial cells have a remarkable plasticity. Recent studies using zebrafish as a model highlight conserved cellular behavior in health and disease in the central nervous system (CNS) between zebrafish and humans. These findings inform our understanding of their function and how their dysregulation in pathogenesis can be determinant
Emerging Role of HDACs in Regeneration and Ageing in the Peripheral Nervous System: Repair Schwann Cells as Pivotal Targets
The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated ax-ons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene ex-pression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.Ministerio de Economía y Competitividad (BFU2016-75864R and PID2019-109762RB-I00)
ISABIAL (UGP18-257 and UGP-2019-128)
Conselleria Educació Generalitat Valenciana (PROMETEO 2018/114)
Medical Research Council (UK) studentship (2251399
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Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell/neuron cocultures.
Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury, used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest, and clear axonal debris
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Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell–neuron cocultures
Peer reviewed: TrueAcknowledgements: We thank Karin Müller, Martin Lenz, and Filomena Gallo at the Cambridge Centre for Advanced Imaging for assistance with microscopy. We thank Aviva Tolkovsky and Alex Clark for technical suggestions. We thank Peter Brophy for the gift of the periaxin antibody. We thank Claire Jacob for helpful suggestions.Funder: Belgian National Fund for Scientific Research F.R.S. - FNRS; doi: http://dx.doi.org/10.13039/501100002661Funder: University of Cambridge; doi: http://dx.doi.org/10.13039/501100000735Myelinating Schwann cell (SC)–dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell–cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris
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Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell-neuron cocultures.
Peer reviewed: TrueAcknowledgements: We thank Karin Müller, Martin Lenz, and Filomena Gallo at the Cambridge Centre for Advanced Imaging for assistance with microscopy. We thank Aviva Tolkovsky and Alex Clark for technical suggestions. We thank Peter Brophy for the gift of the periaxin antibody. We thank Claire Jacob for helpful suggestions.Publication status: PublishedFunder: Belgian National Fund for Scientific Research F.R.S. - FNRS; doi: http://dx.doi.org/10.13039/501100002661Funder: University of Cambridge; doi: http://dx.doi.org/10.13039/501100000735Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris
Emerging role of HDACs in regeneration and ageing in the peripheral nervous system: Repair Schwann cells as pivotal targets
This article belongs to the Special Issue Neuroepigenetic: From Bench to Bedside.The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated axons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene expression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.This work has been funded by grants from the Ministerio de Economía y Competitividad (BFU2016-75864R and PID2019-109762RB-I00), ISABIAL (UGP18-257 and UGP-2019-128) to H. Cabedo and Conselleria Educació Generalitat Valenciana (PROMETEO 2018/114) to H. Cabedo. Medical Research Council (UK) studentship (2251399) to C. Mutschler.Peer reviewe